Learning Target

By the end of this station, the student should be able to:

1. Demonstrate correct handling of serum sample for bilirubin estimation.
2. Interpret serum bilirubin results in relation to jaundice.

Required Material

• Serum sample
• Test tubes / cuvettes
• Pipette or micropipette
• Bilirubin standard
• Diazo reagent
• Accelerator reagent for total bilirubin
• Distilled water / blank
• Colorimeter / spectrophotometer
• Gloves
• Tube rack
• Waste container
• Aluminum foil / dark container for sample protection

Student Task / Procedure

1. Wear gloves and identify the labeled serum sample.
2. Protect the serum sample from direct light.
3. Check that the sample is clear and non-hemolysed.
4. Prepare tubes for Blank, Standard, Total Bilirubin, and Direct Bilirubin.
5. Add serum sample and required reagents according to the method.
6. Add diazo reagent to allow color formation.
7. Use accelerator reagent for total bilirubin estimation.
8. Mix gently and allow reaction time.
9. Read absorbance using colorimeter / spectrophotometer.
10. Calculate bilirubin level and interpret the result in jaundice.

Observation / Identification Points

The student should observe or demonstrate:

• Serum sample is used, not whole blood.
• Sample should be protected from light.
• Hemolysed sample should be avoided.
• Diazo reaction produces colored azobilirubin.
• Direct bilirubin reacts without accelerator.
• Total bilirubin requires accelerator.
• Indirect bilirubin is calculated from total and direct bilirubin.

Result / Interpretation

Principle:
Bilirubin reacts with diazo reagent to form a colored compound called azobilirubin. The intensity of color is proportional to bilirubin concentration.

Important Formula:
Indirect bilirubin = Total bilirubin − Direct bilirubin

Normal Values:

• Total bilirubin: approximately 0.2–1.2 mg/dL
• Direct bilirubin: usually less than 0.3 mg/dL
• Indirect bilirubin: calculated value

Interpretation in Jaundice:

Clinical Significance:
Serum bilirubin estimation helps differentiate hemolytic, hepatic, and obstructive jaundice, which is highly relevant in the GIT module.

Viva Questions

1. Which sample is used for bilirubin estimation?
Answer: Serum sample.

2. Why should bilirubin sample be protected from light?
Answer: Bilirubin is light-sensitive and may degrade, causing falsely low results.

3. What is the principle of bilirubin estimation?
Answer: Bilirubin reacts with diazo reagent to form colored azobilirubin.

4. How is indirect bilirubin calculated?
Answer: Indirect bilirubin = Total bilirubin − Direct bilirubin.

5. Which type of bilirubin is increased in obstructive jaundice?
Answer: Direct / conjugated bilirubin.

Marking Scheme

Total Marks: 5

Common Student Mistakes

• Forgetting to protect the serum sample from light.
• Confusing direct and indirect bilirubin.
• Not linking bilirubin pattern with type of jaundice.

AIM Feedback

Revise this station around three key points: sample handling, diazo reaction, and jaundice interpretation. Always remember that bilirubin is light-sensitive. Direct bilirubin rises mainly in obstructive jaundice, while indirect bilirubin rises mainly in hemolysis.